Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins.

We investigated the properties of ts51, an influenza virus (A/WSN/33) temperature-sensitive RNA segment 7 mutant. Nucleotide sequence analysis revealed that ts51 possesses a single nucleotide mutation, T-261----C, in RNA segment 7, resulting in a single amino acid change. Phenylalanine (position 79) in the wild-type M1 protein was substituted by serine in ts51. This mutation was phenotypically characterized by dramatic nuclear accumulation of the M1 protein and interfered with some steps at the late stage of virus replication, possibly affecting the assembly and/or budding of viral particles. However, although M1 protein was retained within the nucleus, export of the newly synthesized viral ribonucleoprotein containing the minus-strand RNA into the cytoplasm was essentially the same at both permissive and nonpermissive temperatures. The roles of M1 in the export of viral ribonucleoproteins from the nucleus into the cytoplasm and in the virus particle assembly process are discussed.     

Construction and characterization of a bacterial clone containing the hemagglutinin gene of the WSN strain (HON1) of influenza virus

A synthetic dodecadeoxynucleotide primer has been used to prepare a double-stranded DNA form of the hemagglutinin (HA) gene of a human influenza virus (WSN strain, HON1). This DNA has been inserted in plasmid pBR322 and cloned in bacterial cells. The insert contains nearly the complete hemagglutinin gene. A restriction map of this insert has been determined and structurally important areas of the HA gene have been sequenced. Amino acid sequences of several regions of the HA protein were deduced from the DNA sequences and compared to the known amino acid sequences of other influenza A viruses. WSN HA shows extensive homology to all influenza A viruses in a few regions, namely the first 17 amino acids of the N-terminus of HA1 (N-terminal polypeptide of HA) and the first 24 amino acids of the N-terminus of HA2 (C-terminal polypeptide of HA). The sequence diverges extensively from other influenza A viruses in most other areas. The sequence of WSN virus HA is similar to that of other HON1 viruses with the exception of the C-terminus of the HA1 peptide. The change in this area may contribute to some of the unique properties of WSN virus among the HON1 viruses. In addition, WSN HA contains a 17-amino-acid precursor before the N-terminus of HA1 and a single amino acid, arginine, connecting HA1 and HA2.     

Detection of antibodies bound to tumor cell surface antigens with radioiodinated Staphylococcus aureus protein A (SPA)

Summary Staphylococcal protein A (SPA) is known to bind to the Fc portion of certain subclasses of IgG. On the basis of this property, radioiodinated SPA (¹²⁵I-SPA) was used to detect antibodies reacting with surface antigens of tumor cells. Target cells derived from an osteosarcoma growing in C3H/fHeJ mice and antiserum to this tumor prepared in female Hartley guineapigs were used to establish optimum conditions for the assay. Similar optimum conditions were also determined for human melanoma target cells. Target cells were plated at a concentration of either 3×10⁵ cells per well or 1×10⁵ cells per well in Microtest II plates, and allowed to form semiconfluent monolayers for 24–48 h respectively. Target cells thus prepared were treated with antiserum and then with ¹²⁵I-SPA. A minimum of 30 min incubation time was found to be optimal for the antigen-antibody reaction. The quantity of ¹²⁵I-SPA bound to antisera-treated target cells was found to depend on the time of incubation with ¹²⁵I-SPA and on the concentration of SPA used. Longer incubation times and increasing concentrations of SPA resulted in greater amounts of ¹²⁵I-SPA being bound to antiserum treated target cells. This assay was employed for the detection of antibodies in the sera of two melanoma patients and two colon carcinoma patients. The results of absorption analysis suggest that the antibody activity in the sera of the melanoma patients may be of four different specificities: (a) autoantibodies, (b) alloantibodies, (c) antibodies reacting with common, cross-reacting melanoma-associated antigens, and (d) antibodies reacting with unique antigens specific for autologous melanoma cells.     

Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

Although the insertion of heme into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process are not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3-h period and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structures, heme ligation states, and functions (three NO synthases, two cytochrome P450's, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme levels, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process.     

Tunable luminescence of a synthesized furophenanthraquinone derivative: interactions with different solvents

The synthesis is described of a luminescent furophenanthraquinone derivative, 9‐methoxyphenanthro[4,3‐b]furan‐4,5‐dione (MPFD). The biological importance of tetracyclic furophenanthraquinones was considered and the tunable luminescence of MPFD in different solvents was studied to explore the nature of the specific interactions between MPFD and solvents. Observation of dual emission bands and identical nature of the fluorescence excitation spectra of MPFD monitored at the emission wavelength in polar solvents indicated the formation of two different types of species in the excited state, probably due to proton transfer from the solvent to MPFD. Luminescence intensity due to anionic species was found to be increased and the corresponding peak was red shifted with increase in the proton‐donating ability of the solvents, acting as an acid with respect to MPFD. Availability of more acidic protons in the solvent facilitated this phenomenon occurring in the excited state. MPFD also interacted with halogen‐containing solvents by forming electron donor–acceptor charge transfer (CT) complexes. This CT complex formation was dependent on the number of chlorine atoms; the position of the corresponding luminescence band varied with the polarity of the solvent. Extent of the CT increased with increase in the number of chlorine atoms in the dichloro, trichloro and tetrachloro solvents, whereas the luminescence peak due to the CT complex was found to be blue shifted with decrease in solvent polarity. Interaction of the synthesized bioactive MPFD with different solvents deserves biological importance as proton transfer and CT play pivotal roles in biology.     

Design and in silico modeling of Indoloquinoxaline incorporated keratin nanoparticles for modulation of glucose metabolism in 3T3-L1 adipocytes

The following study was done to assess the glucose utilizing efficiency of Indoloquinoxaline derivative incorporated keratin nanoparticles (NPs) in 3T3-L1 adipocytes. Indoloquinoxaline derivative had wide range of biological activities including antidiabetic activity. In this view, Indoloquinoxaline moiety containing N, N-dimethyl (3-fluoro-6H-indolo [3,2-b] quinoxalin-6-yl) methanamine compound was designed and synthesized, and further it is incorporated into keratin nanoparticles. The formulated NPs, drug entrapment efficiency, releasing capacity, stability, and physicochemical properties were characterized by various spectral analyzer and obtained results of characterizations were confirmed the properties of NPs. The analysis of mechanism underlying the glucose utilization of NPs was examined through molecular docking with identified target, and observed in silico study reports shown strong interaction of NPs in the binding pockets of AMPK and PTP1B. Based on the in silico screening, the formulated NPs was performed for in vitro cellular viability and glucose uptake studies on 3T3-L1 adipocytes. Interestingly, 40 μg of NPs displayed 78.2 ± 2.76% cellular viability, and no cell death was observed at lower concentrations. Further, the concentration dependent glucose utilization was observed at different concentrations of NPs in 3T3-L1 adipocytes. The results of NPs (40 μg) on glucose utilization have revealed eminent result 58.56 ± 4.54% compared to that of Metformin (10 μM) and Insulin (10 μM). The identified results clearly indicated that Indoloquinoxaline derivative incorporated keratin NPs significantly increased glucose utilization efficiency and protect the cells against the insulin resistance.     

Efficient decolorization and detoxification of sulphonated azo dye Ponceau 4R by using single and mixed bacterial consortia

Abstract

The decolorization of toxic azo dye Ponceau 4R by three strains of bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1 individually and in consortia was studied. At optimal conditions, up to 95%, 93% and 87% of the dye was decolorized by the strains AK1, AK2 and VKY1, respectively, in 24?h at 200?mg/L of the dye. Decolorization of the dye was optimized for different parameters such as the concentration of dye, pH, temperature and NaCl concentration. These strains were able to decolorize Ponceau 4R up to an initial concentration of 800?mg/L in the pH range of 5–10, temperature 25–55?°C and NaCl concentration up to 30?g/L. The dye decolorization efficiency of these strains was further enhanced by using different consortia of AK1, AK2 and VKY1 in various combinations. The complete decolorization of the dye by a consortium was achieved within 18?h at 200?mg/L. The cell-free extract of these strains grown on this dye exhibited a remarkable activity of azoreductase which is involved in the breakage of the azo bond. The steady-state kinetics of azoreductase, validated the ping pong Bi-Bi mechanism of enzyme action. UV–Vis spectra, HPLC, FTIR and LC-MS analysis of the dye decolorized samples showed the formation of 4-aminonaphthalene-1-sulphonic acid and 5-amino-6-hydroxynaphthalene-2, 4-disulphonic acid as the products of azo bond breakage. The phytotoxicity test of decolorized sample revealed a considerable reduction in the toxicity in comparison with the parent dye.     

Synthesis of a Smart Gold Nano‐vehicle for Liver Specific Drug Delivery

Targeting drug formulations to specific tissues and releasing the bioactive content in response to a certain stimuli remains a significant challenge in the field of biomedical science. We have developed a nanovehicle that can be used to deliver “drugs” to “specific” tissues. For this, we have simultaneously modified the surface of the nanovehicle with “drugs” and “tissue-specific ligands”. The “tissue-specific ligands” will target the nanovehicle to the correct tissue and release the “drug” of interest in response to specific stimuli. We have synthesised a “lactose surface-modified gold nanovehicle” to target liver cells and release the model fluorescent drug (coumarin derivative) in response to the differential glutathione concentration (between blood plasma and liver cells). Lactose is used as the liver-specific targeting ligand given the abundance of l-galactose receptors in hepatic cells. The coumarin derivative is used as a fluorescent tag as well as a linker for the attachment of various biologically relevant molecules. The model delivery system is compatible with a host of different ligands and hence could be used to target other tissues as well in future. The synthesised nanovehicle was found to be non-toxic to cultured human cell lines even at elevated non-physiological concentrations as high as 100 μg/mL. We discover that the synthesised gold-based nanovehicle shows considerable stability at low extracellular glutathione concentrations; however coumarin is selectively released at high hepatic glutathione concentration.     

Smokeless Tobacco Extract (STE)-Induced Toxicity in Mammalian Cells is Mediated by the Disruption of Cellular Microtubule Network: A Key Mechanism of Cytotoxicity

Smokeless tobacco usage is a growing public health problem worldwide. The molecular mechanism(s) underlying smokeless tobacco associated tissue damage remain largely unidentified. In the present study we have tried to explore the effects of aqueous extract of smokeless tobacco (STE) on tubulin-microtubule, the major cytoskeleton protein that maintains cells morphology and participates in cell division. Exposure to STE resulted in dose-dependent cytotoxicity in a variety of mammalian transformed cell lines such as human lung epithelial cells A549, human liver epithelial cells HepG2, and mouse squamous epithelial cells HCC7, as well as non-tumorogenic human peripheral blood mononuclear cells PBMC. Cellular morphology of STE-treated cells was altered and the associated disruption of microtubule network indicates that STE targets tubulin-microtubule system in both cell lines. Furthermore it was also observed that STE-treatment resulted in the selective degradation of cellular tubulin, whereas actin remains unaltered. In vitro, polymerization of purified tubulin was inhibited by STE with the IC₅₀ value∼150 µg/ml and this is associated with the loss of reactive cysteine residues of tubulin. Application of thiol-based antioxidant N-acetyl cysteine (NAC) significantly abrogates STE-mediated microtubule damage and associated cytotoxicity in both A549 and HepG2 cells. These results suggest that microtubule damage is one of the key mechanisms of STE-induced cytotoxity in mammalian cells.